How to make western results clean??? - (Sep/13/2005 )

Hi, everyone, I am doing IP now. In the first try, the film turs out to be very dirty. The places I hope my bands should show up have very strong signal so I don't know whether it is nonspecific bands(such as: heavy or light IGg, as my protein is around 53KDa). In addition, there are many nonspecific bands show up at other places. Now I am not sure how to make the film a liittle clean. I don't know whether I should decrease the amount of lysate loading on the gel, increase the dilution number of the first AT or increase the dilution number of the second AT. As to dilution number of first AT, it is 1:1000 now.The instruction says for western the dilution is 1:1000. So I am wondering if I make it more diluted, will the AT still work?

Could anyone give any idea?

Thank you very much!!!!

-popogirlxd-

I think, that you can really dilute your Ab. Also to get rid of background, you can prolongate blocking step, try different blocking agents ( milk, BSA), and shorten the incubation time with Ab

For example, me, I did the solutions of Abs in BSA and re-use them in Westerns million times, until I saw that nearly all Ab was titrated from soultion , and I get fade band, or higher than usual background

-Alesia-

QUOTE (Alesia @ Sep 14 2005, 02:29 AM)

I think, that you can really dilute your Ab. Also to get rid of background, you can prolongate blocking step, try different blocking agents ( milk, BSA), and shorten the incubation time with Ab

For example, me, I did the solutions of Abs in BSA and re-use them in Westerns million times, until I saw that nearly all Ab was titrated from soultion , and I get fade band, or higher than usual background

So you mean I can dilute the fist AT , even the company says do 1:1000 dilution in western , right?

Thanks

-popogirlxd-

Yes you can dilute it more. In my experience the company always suggests a dilution that is way too high, we routinely start at least 10X more diluted than company suggests...